In microscopic examinations and documentation, it often happens that multiple exposures and assembled images that depict different aspects of the specimen are produced. The images thus produced are generated using various contrasting methods or various microscopy methods, and then combined into one image. The various image constituents are then displayed, for example, using so-called “false colors.” For example, the samples to be examined are often marked with various fluorescent dyes that can be excited and observed using different filter combinations. The images achieved in this fashion, each produced with a specific filter combination, are subsequently assembled into a single cumulative image.
The case considered here refers to the combination of images acquired by means of interference contrast transmitted-light (ICT) microscopy and fluorescence microscopy. The image generated with the ICT method then shows the unstained biological specimen as a whole. The image produced by means of fluorescence microscopy, on the other hand, shows only particular and specific markings at certain points. The images produced with the two methods are then assembled into one overlay image.
The overlay images produced in this fashion are each imaged onto a camera. It is desirable, in producing an overlay image of this kind, for the ICT image to be located on the CCD chip of the camera at exactly the same point as the fluorescence images or a bright-field transmitted-light image. The reason is that if the images are imaged onto the CCD chip with an offset from one another, evaluation of the image is negatively affected.
In the ICT method, a polarizing filter that serves as the analyzer is located in the imaging beam path of the microscope. This analyzer is usually arranged between the microscope objective and the tube lens. Since the analyzer has a transmissivity of only approx. 30% for unpolarized light, it is also usually introduced into the beam path only for the ICT method. For the fluorescence measurement or fluorescence observation, in which weak intensities generally occur, the analyzer is removed from the beam path.
Polarizing filters of planar configuration, such as those used in microscopy, usually comprise a stretched polarizing film that is cemented between two glass plates. Such polarizing filters have the disadvantage that incident light is slightly deflected. The reasons for this beam deflection include the stretched polarizing film itself, wedge-shaped cemented surfaces, and possibly wedge-shaped glass plates. Known polarizing filters of this type thus produce beam deflections of up to 3 minutes. If the microscope has, for example, a tube lens with a focal length of 200 millimeters, an analyzer of this kind with a beam deflection of 3 minutes produces approximately a 175-μm offset of the intermediate image of the sample, With a CCD camera having a pixel size of approx. 8 μm, the result is that the analyzer causes the image to be offset by approximately 20 pixels.
Until now, the offset produced on the CCD chip between the ICT image and the fluorescence image had to be tolerated, meaning that the associated loss in the quality of the overlay images necessarily had to be accepted. Alternatively, the image offset of the individual images in the overlay image was compensated for using software in the context of digital image acquisition or the processing of the overlay images, which required appropriate software and hardware and is time-consuming.